Hair analysis plays an important role in abstinence control in forensic toxicology. However, hair coloration affects the concentrations of xenobiotics and may lead to false negative results. For instance, henna has been shown to decrease ethyl glucuronide concentrations in hair. For analysis of the main henna ingredient lawsone (2-hydroxy-1,4-naphthoquinone), hair samples were washed, cut into small pieces (less than 5 mm), incubated (20 mg) in water and with internal standard (theophylline) for 24 h at 37 °C. Lawsone was analyzed in the supernatant using liquid chromatography with diode array detection. For quantitative assay, the absorption at 280 nm was found to be linear up to 250 ng/mg hair. The detection limit of lawsone was 2.2 ng/mg, precision and accuracy were better than 6%. Lawsone was only detectable in 12 henna-colored hair samples in concentrations from 27.3 to 253.7 (median 92.6) ng/mg. The analysis of lawsone is recommended in cases of suspected hair coloration where assessment of oxidative treatment was negative (e.g. no increase of 1H-pyrrole-2,3,5-tricarboxylic acid (PTCA) and unobtrusive fluorescence microscopy).